The Flavivirus genus consists of 70 serologically cross-reactive, closely related human or veterinary pathogens causing many serious illnesses, which includes dengue fever, Japanese encephalitis (JE), tick-borne encephalitis (TBE) and yellow fever (YF). The Flaviviruses are spherical with 40-60 nm in diameter with an icosahedral capsid which contains a single positive-stranded RNA molecule.
YF virus is the prototype virus of the family of the Flaviviruses with a RNA genome of 10,862 nucleotides (nt), having a 5' CAP structure (118 nt) and a nonpolyadenylated 3' end (511 nt). The complete nucleotide sequence, of its RNA genome was determined by Rice, C. M. et al (1985).
The single RNA is also the viral message and its translation in the infected cell results in the synthesis of a polyprotein precursor of 3,411 amino acids which is cleaved by proteolytic processing to generate 10 virus-specific polypeptides. From the 5' terminus, the order of the encoded proteins is: C; prM/M; E; NS1; NS2A; NS2B; NS3; NS4A; NS4B and NS5. The first 3 proteins constitute the structural proteins, i.e., they form the virus together with the packaged RNA molecule. The remainder of the genome codes for the nonstructural proteins (NS) numbered from 1 through 5, according the order of their synthesis.
The C protein, named capsid, has a molecular weight ranging from 12 to 14 kDa (12-14 kilodaitons); the membrane protein, M has a molecular weight of 8 kDa, and its precursor (prM) 18-22 kDa; the envelope protein, E, has 52-54 kDa, being all of them encoded in the first quarter of their genome.
Three of the nonstructural proteins are large and have highly conserved sequences among the flaviviruses, namely, NS1 has a molecular weight ranging from 38 to 41 kDa; NS3 has 68-70 kDa and NS100-103 kDa. No role has yet been assigned to NS1 but NS3 has been shown to be bifunctional having a protease activity needed for the processing of the polyprotein, and the other is a nucleotide triphosphatase/helicase activity which is associated with viral RNA replication. NS5, the largest and most conserved protein, contains several sequence motifs which are characteristic of viral RNA polymerases. The 4 small proteins , namely NS2A, NS2B, NS4A and NS4B, are poorly conserved in their amino acid sequences but not in their pattern of multiple hydrophobic stretches. NS2A has been shown to be required for proper processing of NS1 whereas NS2B has been shown to be associated with the protease activity of NS3.
Two strains of yellow fever virus (YF), isolated in 1927, gave rise to the vaccines to be used for human immunization. One, the Asibi strain, was isolated from a young african named Asibi by passage in Rhesus monkey (Macaca mulatta), and the other, the French Viscerotropic Virus (FVV), from a patient in Senegal.
In 1935, the Asibi strain was adapted to growth in mouse embryonic tissue. After 17 passages, the virus, named 17D, was further cultivated until passage 58 in whole chicken embryonic tissue and thereafter, until passage 114, in denervated chicken embryonic tissue only.
Theiler and Smith (Theiler, M. and Smith, H. H. (1937). "The effect of prolonged cultivation in vitro upon the pathogenicity of yellow fever virus". J. Exp. Med. 65:767-786) showed that, at this stage, there was a marked reduction in viral viscero and neurotropism when inoculated intracerebrally in monkeys. This virus was further subcultured until passages 227 and 229 and the resulting viruses, without human immune serum, were used to immunize 8 human volunteers with satisfactory results, as shown by the absence of adverse reactions and seroconversion to YF in 2 weeks. These passages yielded the parent 17D strain at passage level 180 (see FIG. 1), 17D at passage 195, and the 17D-204 at passage 204. was further subcultured until passage 241 and underwent 43 additional passages in embryonated chicken eggs to yield the virus currently used for human vaccination in some countries (passage 284). The 17D-204 was further subcultured to produce Colombia 88 strain which, upon passage in embryonated chicken eggs, gave rise to different vaccine seed lots currently in use in France (I. Pasteur, at passage 235) and in the United States (Connaught, at passage 234). The 17D-213 strain was derived from 17D-204 when the primary seed lot (S1 112-6-9) from the Federal Republic of Germany (FRG 83-66) was used by the World Health Organization (WHO) to produce an avian leukosis virus-free 17D seed (S1 213/77) at passage 237.
In the late 1930's and early 1940's, mass vaccination was conducted in Brazil with the use of several substrains of 17D virus (Table I). These substrains differed in their passage history and they overlapped with regard to time of their use for inocula and/or vaccine production. The substitution of each one by the next was according to the experience gained during vaccine production, quality control and human vaccination in which the appearance of symptomatology led to the discontinuation of a given strain.
Each of these 17D-204 strains (C-204; F-204) was plaque purified in different cell lines, the virus finally amplified in SW13 cells and used for CDNA cloning and sequence analyses (Rice, C. M.; Lenches, E.; Eddy, S. R.; Shin, S. J.; Sheets, R. L. and Strauss, J. H. (1985). "Nucleotide sequence of yellow fever virus: implications for flavivirus gene expression and evolution". Science. 229: 726-733; Despres, P.; Cahour, A.; Dupuy, A.; Deubel, V.; Bouloy, M.; Digoutte, J. P.; Girard, M. (1987). "High genetic stability of the coding region for the structural proteins of yellow fever strain 17D". J. Gen. Virol. 68: 2245-2247).
The 17D-213 at passage 239 was tested for monkey neurovirulence (R. S. Marchevsky, personal communication, see Duarte dos Santos et al, 1995) and was the subject of sequence analysis together with 17DD (at passage 284) and comparison to previously published nucleotide sequences of other YF virus strains (Duarte dos Santos et al, 1995) (Asibi: Hahn, C. S.; Dalrymple, J. M.; Strauss, J. H. and Rice, C. M. (1987). "Comparison of the virulent Asibi strain of yellow fever virus with the 17D vaccine strain derived from it". Proc. Natl. Acad. Sci. USA. 84: 2029-2033; 17D-204 strain, C-204: Rice. C. M.; Lenches, E. M.;
Eddy, S. R.; Shin, S. J.; Sheets, R. L. and Strauss, J. H. (1985). "Nucleotide sequence of yellow fever virus: implications for flavivirus gene expression and evolution". Science. 229: 726-733; F-204: Despres, P.; Cahour, R.; Dupuy, A.; Deubel, V.; Bouloy, M.; Digoutte, J. P. and Girard, M. (1987). "High genetic stability of the coding region for the structural proteins of yellow fever virus strain 17D". J. Gen. Virol. 68: 2245-2247) (see FIG. 1).
A total of 67 nucleotide differences, corresponding to 31 amino acid changes, were originally noted between the Asibi and 17D-204 genomic sequences (see Hahn, C. S. et al, 1987). The comparison between the nucleotide sequences of 17DD and 17D-213 substrains (see Duarte dos Santos et al, 1995) and the nucleotide sequence of 17D-204 substrain (see Rice et al, 1985) showed that not all chances are common and thus not confirmed as being 17D-specific. Therefore, the 17D-substrain specific changes observed are very likely not related to attenuation but may reflect differences in behavior of these strains in monkey neurovirulence tests. In consequence, the number of changes likely to be associated with viral attenuation were reduced by 26%, i.e., to 48 nucleotide changes. From these 48 nucleotide sequence changes which are scattered along the genome, 26 are silent mutations and 22 led to amino acid substitutions. More important are the alterations noted in the E protein because it is the main target for humoral neutralizing response, i.e., it is the protein where hemagglutination and neutralization epitopes are located, and it mediates cell receptor recognition and cell penetration, therefore targeting the virus to specific cells. Importantly, E protein accumulate the highest ratio of nonconservative to conservative amino acid changes. Altogether, eleven nucleotide substitutions were observed in the E protein gene leading to 8 amino acid changes at positions 52, 170, 173, 200, 299, 305, 331 and 380 (respectively nucleotides 1127, 1482, 1491, 1572, 1870, 1887, 1965 and 2112 from the RNA 5' end).
Alterations at amino acids 52 and 200 are located in domain A of E protein (domain II in 3-D structure proposed for Flaviviruses E protein--Rey, F. A.; Heinz F. X.; Mandl, C.; Kunz, C and Harrison, S. C. (1995). "The envelope glycoprotein from tick-borne encephalitis virus at 2 .ANG. resolution". Nature. 375: 291-298) which is conserved among Flaviviruses and contains cross-reactive epitopes as shown by Mandl, C. W. et al (Mandl, M. W.; Guirakhoo, F.; Holzmann, H.; Heinz, F. X. and Kunz, C. (1989). "Antigenic structure of the flavivirus envelope E protein at the molecular level using tick-borne encephalitis virus as a model". J. Virol. 63: 564-571). This domain II is highly crosslinked by disulphide bonds and undergoes low pH transition which is related to exposing a strictly conserved and hydrophobic stretch of amino acids which are supposed to be involved in the fusion of the viral envelope to the endosome membrane.
Alterations at amino acids 299, 305, 331 and 380 are located in the B domain (domain III in the 3-D structure--see Rey, F. A. et al). This domain was suggested to be involved in viral attachment to a cellular receptor and consequently being a major determinant both of host range and cell tropism and of virulence/attenuation. The 4 amino acid changes reported for YF are located on the distal face of domain III. This area has a loop which is a tight turn in tick-borne encephalitis virus but contains 4 additional residues in all mosquito-borne strains. Because viruses replicate in their vectors, this loop is likely to be a host range determinant. This enlarged loop contains an Arginine-Glycine-Aspartic Acid (Arg-Gly-Asp) sequence in all 3 YF 17D vaccine strains. This sequence motif is known to mediate a number of cell interactions including receptor binding and is absent not only in the parental virulent Asibi strain but also in other 22 strains of YF wild type virus (Lepiniec, L.; Dalgarno, L.; Huong, V. T. Q.; Monath, T. P.; Digoutte, J. P. and Deubel, V. (1994). "Geographic distribution and evolution of yellow fever viruses based on direct sequencing of genomic DNA fragments". J. Gen. Virol. 75: 417-4123). Such a fact suggests that the mutation from Threonine (Thr) to Arginine (Arg), creating a Arg-Gly-Asp motif, is likely to be relevant for the attenuated phenotype of the YF 17D strain. Consistently, Lobigs et al (Lobigs, M.; Usha, R.; Nesterowicz, A.; Marschall, I. D.; Weir, R. C. and Dalgarno, L. (1990). "Host cell selection of Murray Valley encephalitis virus variants altered at an RGD sequence in the envelope protein and in mouse neurovirulence". Virology. 176: 587-595) identified a Arg-Gly-Asp sequence motif (at amino acid 390) which led to the loss of virulence of Murray Valley encephalitis virus for mice.
Alterations at amino acids 170 and 173 in domain C (domain I of the E protein in the 3-D structure) map very close to the position that a neutralization epitope was identified for tick-borne encephalitis (TBE) virus (see Mandl, C. W. et al) . A mutation at position 171 of TBE virus E protein was shown to affect the threshold of fusion-activating conformational change of this protein and the 2 changes observed for YF 17D virus may be related to same phenomenon. It is conceivable that a slower rate of fusion may delay the extent of virus production and thereby lead to a milder infection of the host. It is noteworthy that the recent development of infectious cDNA for Japanese encephalitis (JE) virus made by Sumiyoshi, H. et al (Sumiyoshi, H.; Hoke, C. H. and Trert, D. W. (1992). "Infectious Japanese encephalitis virus RNA can be synthesized from in vitro-ligated cDNA templates". J. Virol. 66: 5425-5431) allowed the identification of a mutation (Lys for Glu) at amino acid 136 of the E protein which resulted in the loss of neurovirulence for mice (see Sumiyoshi, H.; Tignor, G. H. and Shope, R. E. (1996). "Characterization of a highly attenuated Japanese encephalitis virus generated from molecularly cloned cDNA". J. Infect. Dis. 171: 1144-1151). This means that domain I is an important area which contains a critical determinant of JE virus virulence in contrast to most of the data obtained from the analyses of virulence for several other flaviviruses for which it is suggested that domain III would be the primary site for virulence/attenuation determinants. Nevertheless, such analyses of the E protein provides a framework for understanding several aspects of flavivirus biology and suggests that it should be possible to engineer viruses for the development of new live flavivirus vaccine.
The issue of virulence/attenuation is of special interest for vaccine development but conceivably viral attenuation can result from genetic modification in one or more viral functions. YF virus is the ideal system to study flavivirus virulence and attenuation because: (i) there is a virulent strain (Asibi) from which an extremely well characterized vaccine strain was derived (17D) and has been successfully used for human vaccination for over 50 years; (ii) there is an animal system which reflects human infection; (iii) the complete nucleotide sequences from both virulent and attenuated strains have been determined and (iv) cDNA clones from which infectious RNA can be synthesized are available.
Holland, J. et al (Holland, J.; Spindler, K.; Horodyski, H.; Grabau, E.; Nichol, S. and VandePol, S. (1982). "Rapid evolution of RNA genomes". Science. 215: 1577-1585) described the fact that viral RNA genomes evolve rapidly. Therefore, a given viral population including YF vaccine viruses is likely to consist of a major type sequence population in which genetic variants can be detected. For YF 17D virus, this is easily seen when the virus is plaqued on cultured cells under an semi-solid overlay in which plaques of different sizes are observed. Previous genomic variability analysis using oligonucleotide fingerprinting suggested a high degree of genetic similarity between vaccines produced worldwide with an estimated sequence homology of 98-100%. However, genetic changes were detected and may have occurred within 1-2 passages possibly due to the selection of virion subpopulations or to point mutations. It is unknown whether the outstanding vaccine properties of the YF 17D virus are due to the existence of genetic variants in the vaccine population. Anywise, the stabilization of the YF 17D genome as DNA not only will reduce the accumulation of mutations in the viral genome as seed lots are produced to replace the previous one but will also provide a much more homogeneous population in terms of nucleotide sequence and consequently in terms of phenotypic markers including attenuation for humans, thereby providing the necessary standardization of YF substrain use for vaccine production.
The capability to manipulate the genome of flaviviruses through infectious clone technology has opened new possibilities for vaccine development. This is so because virus can be recovered from complementary DNA by in vitro transcription and transfection of cultured cells with RNA, and these cDNAs corresponding to the complete viral genome allow introducing genetic modifications at any particular site of the viral genome. The pioneer study of Racaniello and Baltimore (Racaniello, V. R. and Baltimore, D. (1981). "Cloned poliovirus complementary DNA is infectious in mammalian cells". Science. 214: 916-919) first showed the feasibility to regenerate virus from cloned cDNA. In the patent U.S. Pat. No. 4,719,177, Racaniello and Baltimore described, in details, the production of RNA viral cDNA by reverse transcribing viral RNA and inserting the resulting cDNA molecule into a recombinant DNA vector. The process was particularly concerned to the production of poliovirus double-stranded complementary DNA (ds cDNA). They found out that the transfected full-length poliovirus cDNA was itself infectious.
In addition, with the development of in vitro transcription systems (see Melton, D. A.; Krieg, P. A.; Rabagliati, M. R.; Maniatis, T.; Zinn, K. and Green, M. R. (1984). "Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter". Nucl. Acids. Res. 12: 7035-7056), a much higher efficiency in the synthesis of full length viral RNA, as compared to cDNA transcription in the cell, became possible. Furthermore, the development of improved transfection methodologies such as cationic liposomes and electroporation increased the efficiency of RNA transfection of cultured cells.
The construction and cloning of a stable full-length dengue cDNA copy in a strain of Escherichia coli using the pBR322 plasmid vector was described by Lai, C. J. et al (Lai, C. J.; Zhao, B.; Hori, H. and Bray, M. (1991). "Infectious RNA transcribed from stably cloned full-length cDNA of dengue type 4 virus". Proc. Natl. Acad. Sci. USA. 88: 5139-5143). They verified that RNA molecules produced by in vitro transcription of the full-length cloned DNA template were infectious, and progeny virus recovered from transfected cells was indistinguishable from the parental virus from which the cDNA clone was derived. But, as mentioned in the Patent Application WO 93/06214, such an infectious DNA construct and RNA transcripts generated therefrom were pathogenic, and that the attenuated dengue viruses generated thus far were genetically unstable and had the potential to revert back to a pathogenic form overtime. To solve this problem, the Applicant proposed to construct cDNA sequences encoding the RNA transcripts to direct the production of chimeric dengue viruses incorporating mutations to recombinant DNA fragments generated therefrom. A preferred mutation ablates NS1 protein glycosylation.
The construction of full-length YF 17D cDNA template that can be transcribed in vitro to yield infectious YF virus RNA was described by Rice et al (Rice, C. M.; Grakoud, A.; Galler, R. and Chambers, T. (1989). "Transcription of infectious yellow fever RNA from full-length cDNA templates produced by in vitro ligation". The New Biologist. 1: 285-296). Because of the instability of full-Length YF cDNA clones and their toxic effects on Escherichia coli, they developed a strategy in which full-length templates for transcription were constructed by in vitro ligation of appropriate restriction fragments. Moreover, they found that the YF virus recovered from cDNA was indistinguishable from the parental virus by several criteria. The YF infectious cDNA is derived from the 17D-204 substrain.
Notwithstanding the YF virus generated from the known YF infectious cDNA is rather attenuated, it cannot be used for human vaccination because of its residual neurovirulence, as determined by Marchevsky, R. S. et al (Marchevsky, R. S.; Mariano, J.; Ferreira, V. S.; Almeida, E.; Cerqueira, M. J.; Carvalho, R.; Pissurno, J. W.; Travassos da Rosa, A. P. A.; Simoes, M. C.; Santos, C. N. D.; Ferreira, I. I.; Muylaert, I. R.; Mann, G. F.; Rice, C. M. and Galler, R. (1995). "Phenotypic analysis of yellow fever virus derived from complementary DNA". Am. J. Trop. Mect. Hyg. 52(1): 75-80).
In short, to obtain a YF vaccine virus using recombinant DNA techniques, it is necessary, cumulatively:
(1) to genetically modify the existing YF infectious cDNA; PA1 (2) to assure that the infectious DNA construct and RNA transcripts generated therefrom give rise to virus which is not pathogenic, and, moreover, does not have the potential to revert to a pathogenic form; PA1 (3) the YF virus generated from cloned cDNA, in addition to being attenuated should retain its immunological properties.
Accordingly, an improved YF virus vaccine without neurovirulence and immunogenic generated from a cloned YF infectious cDNA should be developed for human immunization.